(1) Field of the Invention
The present invntion relates to a potential-causing membrane for use as an immunosensor, i.e., a receptor which can identify an antigen or an antibody to be determined on the surface thereof and which can directly detect an immuno reaction occurring on the surface as an electrical current or voltage.
(2) Prior Art
In the field of clinical medical science, recently use of an immuno reaction, i.e., an antigen-antibody reaction, has come into general use to diagnose, prevent or cure diseases. Regarding clinic test methods, there have also been established various methods such as use of precipitation-agglutination reaction, fluorescent antibody technique, radioimmunoassay, enzyme-immunoassay and the like. However, since these conventional methods have some disadvantages such as that they require expensive equipment and sophisticated techniques to carry out the tests, such methods are very difficult to carry out on an everyday basis for qualitatively or quantitatively detection of biological substances, for example, in the home.
Nowadays, there are proposed new methods of detecting biological substances, which utilize antigen-antibody reaction carried out on the surface of a thin membrane, or on the interface of some solids. For example, Giaever et. al, found out that, when an antigen-antibody reaction is carried out on thin film comprising fine particles of indium coated on a glass plate, the degree of light transmission of the film greatly increases (J. Immunology, 110 (1973), 1424). Del Castillo et. al. reported that electrical resistance of lipid bimolecular membrane decreases when antigen-antibody reaction is carried out on the membrane (Science 153 (1966), 183). Suzuki, Aizawa, et. al. also reported fixing an antigen or an antibody is on a cellulose acetate membrane and determining the electrical potential of the membrane so as to detect biological materials (J. Membrance Sci., 2 (1977), 125 etc.). Furthermore, Janata showed that using concanavallin A as a substance similar to an immune fixed on a thin film formed of a polymer coated on the surface of a platinum electrode, it is possible to detect polysaccharides which specifically bond with concanavallin A by measuring the change in electric potential (J. Am. Chem. Soc., 97 (1935), 2914 ). Yamamoto, Tsubomura, et. al., proposed that it is possible to detect biological substances by measuring the change in electric potential between electrodes due to immune reaction, using electrodes comprising a chemically modified antigen or antibody (J. Chem. Soc. (1980), 1562). Thus, there have been proposed many immunosensors based on the clever utilization of both functions of identifying and binding antigen utilizing antibodies.
As described above, the immunosensor is based on the principle of immunoassay and is generally classified into marking immunoassay using a marker and non-marking immunoassay which does not use a marker. Regarding marking immunoassay, there has been invented a good determination device in which enzymes, metal chelate compounds, red blood cells, liposomes and the like are used as a marker and the respective final change is transformed into an electric signal by an electrochemical transducer. With the immunosensor, an extremely high sensitivity can be obtained by using chemical amplification.
On the other hand, with non-marking immunoassay, an antigen-antibody complex is formed on the surface of the receptor and the resulting physical change is directly transformed into an electric signal. So far there have been proposed two kinds of non-marking immunoassay, one being a membrane potential method in which a receptor is formed by binding an antibody (or antigen) onto the surface of the membrane and measuring the electric potential before and after the antigen-antibody reaction, and the other being an electrode potential method in which a receptor is formed by binding antibody (or antigen) directly or through membrane onto the surface of an electrode, and measuring the change in electrode potential caused by the antigen-antibody reaction. In this connection, the membrane or antibody bound (or fixed) on the surface of the electrode forms a stable complex by reacting with the antigen. The membrane or the antigen-antibody complex formed on the surface of the electrode induces a change in membrane potential or electrode potential.
An immunosensor which determines electrical potential based on the above-described principle is a new method that is recently attracting attention and which is being studied, since antigens and antibodies can be directly and easily detected according to the method. However, since the response time of a non-marking immunosensor of either of the above two kinds is long (not less than 30 minutes) and the electrical sensitivity of the response is also low, the properties of the non-marking immunosensor are not sufficient. It is therefore necessary to improve the properties of the immunosensor in practical use.
In the immunosensor as described above, there are some cases where a receptor of the electrode potential type is formed by binding antigen (or antibody) directly onto the surface of the electrode, but as a rule, a receptor, i.e., a potential-causing membrane of the above two types, is formed by chemically or physically fixing an antigen (or antibody) onto the surface of a membrane of a molecular compound such as cellulose acetate, sephadex, polystyrene, polyamide or the like. In this regard, it is estimated that electrical response of the membrane mainly depends on the amount of antigen (or antibody) which is fixed onto the membrane, an depends on the electrical conductivity of the membrane perse. The membrane perse generally has insulative properties.